Method for treating a disease or condition responsive to opening of c1c-2 channel

ABSTRACT

Disclosed is a method for prophylactic or therapeutic treatment of a condition or disease responsive to opening of ClC-2 channel, which comprises the step of administrating an effective amount of a ClC-2 channel opener to a subject in need of said treatment. According to the invention, a tissue or organ to be transplanted can also be treated with the ClC-2 channel opener.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 10/298,062, filed Nov.18, 2002, which claims benefit of Provisional Application No. 60/331,542filed Nov. 19, 2001. The entire disclosures of the prior applicationsare incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to a method for treating a disease or acondition responsive to opening of ClC-2 channel.

BACKGROUND ART

A chloride channel is an ion-transport membrane protein for transportingchloride ions (Cl⁻). It has been reported that various kinds of chloridechannels are present in the cell membrane of nerve, muscle andepithelium, and they are involved with various physiological functionsand cytophylaxis mechanisms.

For example, a chloride channel cloned from crampfish's electric organand named ClC-O was later found to form a large family (ClC family).Examples of ClC family are: ClC-1 present in the skeletal muscle ofmammals; ClC-2 present in the epithelium of various organs; ClC-3 andClC-4 distributed in hippocampus, cerebellum, etc.; ClC-5 present inlung, kidney, etc.; ClC-6 and ClC-7 present in brain, testis, skeletalmuscle, kidney, etc.; and ClCK-1 and ClCK-2 specifically shown only inkidney.

It is generally said that various channels are involved with a number ofdiseases. It is generally believed that the dysfunctioning of thesechannels or the existence of regulation defects in processes thatactivate such channels may play an important role in the pathogenesis ofsuch diseases and illnesses. As a result, a compound, which opensvarious channels and assists regulation of electrophysiological functionof cells, could have important therapeutic and prophylactic abilitiesfor treatment and relief of such conditions.

It is inferred that a ClC-2 channel therapeutically plays an importantrole in cystic fibrosis, which is an autosomal recessive inheriteddisease best known in the Caucasian race (Cuppaletti et al., AmericanJournal of Physiology. Cell Physiology, 281(1), C46-54, 2001; Joo etal., Biochemica et Biophysica Acta, 1446(3), 431-437, 1999 andSchwiebert et al., Proc. Natl. Acad. Sci. USA 97(7), 3879-3884, 1998).However, the details of other roles are not known very well.

SUMMARY OF THE INVENTION

The present inventor has conducted intensive studies using a compoundhas an ability to open ClC-2 channel and found that a ClC-2 channelopener is effective for treatment of various diseases.

Namely, the present invention relates to a method for prophylactic ortherapeutic treatment of a condition or disease responsive to opening ofClC-2 channel, which comprises administrating an effective amount of aClC-2 channel opener to a subject in need of said treatment, providedthat the disease is not cystic fibrosis. The present invention furtherrelates to a method for treatment of a tissue or organ to betransplanted in a transplantation process, comprising the step ofcontacting the tissue or organ with a pharmaceutical compositioncomprising a ClC-2 channel opener as an active ingredient.

Further, the present invention also relates to a pharmaceuticalcomposition for prophylactic or therapeutic treatment of a condition ordisease responsive to opening of ClC-2 channel, which comprises a ClC-2channel opener as an active ingredient, provided that the disease orcondition is not cystic fibrosis. The present invention also relates toa pharmaceutical composition for treating a tissue or organ to betransplanted in a transplantation procedure, which comprises a ClC-2channel opener as an active ingredient.

Furthermore, the present invention relates to use of a ClC-2 channelopener for manufacturing a pharmaceutical composition for prophylacticor therapeutic treatment of a condition or disease responsive to openingof ClC-2 channel, provided that the disease is not cystic fibrosis, Thepresent invention also relates to use of a ClC-2 channel opener formanufacturing a pharmaceutical composition for treatment of a tissue ororgan to be transplanted in a transplantation procedure.

DETAILED DESCRIPTION OF THE INVENTION

The ClC-2 channel opener used in the present invention is notparticularly limited and may be any compound as far as it has a ClC-2channel opening activity. The ClC-2 channel opening activity may beconfirmed by measuring the increase of chloride-ion flows through aClC-2 channel in a cell membrane from inside to outside of the cell orin the opposite direction. For instance, it is possible to carry out ascreening for a compound having ClC-2 channel opening activity by usinga known assay strategy such as patch clamp method.

Examples of compounds having ClC-2 channel opening activity includecyclooxygenase inhibitor or nonsteroidal anti-inflammatory agent such asibuprofen and ebselen, protein kinase A, oleic acid, elaidic acid,arachidonic acid, cell growth factor, such as TGF_(α) (transforminggrowth factor-α) and KGF (keratinocyte growth factor)), prostaglandincompound and benzimidazole derivative.

In the present invention, “a disease or a condition responsive toopening of ClC-2 channel” includes a condition, morbidity, disease anddisorder prophylactically or therapeutically responsive or sensitive toactivation or opening of ClC-2 channel in the cell of target tissues;and a condition, morbidity, disease and disorder prophylactically ortherapeutically relate to ClC-2 channel behavior. As far as they arediseases or conditions responsive to ClC-2 channel opening, other thancystic fibrosis, there is no limitation. Examples of said diseases orconditions include hepatic disease such as hepatitis and cirrhosis,pancreatic disease such as pancreatitis, constipation, exocrine disordersuch as dry eye and dry mouth, cerebral function disorder,cardiovascular system disorder, respiratory system disorder anddigestive system disorder.

Accordingly, “subject in need of said treatment” may be a subject who issuffering from above-discussed condition or disease, or a subject who issusceptible to suffering from such condition or disease. The subject maybe any mammalian subject including human beings.

According to the invention, a pharmaceutical composition comprising acompound having ClC-2 channel opening activity is administrated to apatient in need of such treatment. The pharmaceutical composition may bein a form suitable for systemic or topical application, which is to beadministrated by oral, intravenous (including drip infusion),subcutaneous, intrarectal, intravaginal, percutaneous or ocularadministration. Examples of dosage forms used in the instant inventioncomprise, but not limited to, internal preparation, injection, drips,external preparation, eye drops, suppository and pessary.

The dose of the active ingredient may vary according to the type of thesubject such as animals or human, age, weight, symptom to be treated,desirable therapeutic effect, administration route and period fortreatment, and a suitable dose may be chosen according to the compoundto be used.

The pharmaceutical composition of the invention may further comprisephysiologically acceptable, suitable additives in addition to the activeingredient. The term additives” used herein is the generic name for thefollowing: excipient, diluent, filler, resolvent, lubricant, adjuvant,binder, disintegrator, coating agent, encapsulating agent, ointmentbase, suppository base, aerosol, emulsifier, dispersant, suspension,thickener, isotonizing agent, buffer, soothing agent, preservative,antioxidant, corrigent, flavor, colorant and functional material such ascyclodextrin and biodegrading polymer. These additives are well known tothe art and may be selected as desired based on descriptions in generalbooks on pharmaceutics.

The term “treatment” used herein includes any means of control such asprevention, care, relief of the condition, attenuation of the conditionand arrest of progression.

According to the present invention, the pharmaceutical compositioncomprising a ClC-2 channel opener as an active ingredient is effectivefor treatment of a disease or a condition responsive to opening of ClC-2channel. Examples of the disease or condition may include hepaticdisease, pancreatic disease, constipation, exocrine disorder, cerebralfunction disorder, cardiovascular system disorder, respiratory systemdisorder and digestive system disorder. Especially, the composition hasan ability to promote secretion of chloride ion in organs that controlfluid secretion. Said promotion of chloride ion secretion results in,for example, acceleration of bile secretion in liver, acceleration ofintestinal fluid secretion in bowel, and acceleration of lacrimation andsalivary secretions in exocrine system such as the lacrimal and salivaryglands. Accordingly, the pharmaceutical composition of the invention iseffective for treatment of hepatic disease, exocrine disorder such asdry eye and dry mouth and constipation.

The composition or method of the present invention is also effective fortreatment of a tissue or organ to be transplanted in a transplantationprocedure. By treating the tissue or organ with the composition of theinvention, organ hypoactivity as well as concomitant disease, which mayoccur during or after the transplantation operation, are effectivelyprevented.

In the transplantation procedure, it is necessary not only to maintainthe function of the tissue or the organ for a certain period until theyare grafted to the recipient but also to ensure the tissue is well takenby the recipient after the surgery. The composition of the presentinvention may be used for treatment of a tissue or organ upon removal ofthe tissue or organ from the donor, for preservation and maintenance ofthe removed tissue or organ, for treatment of the tissue or organ aswell as the recipient upon grafting surgery, and for post-operationaltreatment.

During the transplantation process, the pharmaceutical composition ofthe present invention may be administrated in vivo to the donor orapplied directly to the removed tissues or organs. For example, thecomposition may be used as a perfusate for in vivo perfusion of thedonor at the removal operation, as a perfusate for ex vivo perfusion ofthe removed organ, as a preservative solution for the removed organ andas a rinse solution before blood reperfusion at grafting the organ tothe recipient. The composition of the invention may be employed aseither one of perfusate, preservative solution and rinse solution, ortwo or all of these solutions.

According to the present invention, the composition may contain a singleactive ingredient or a combination of two or more active ingredients. Ina combination of plural active ingredients, their respective dose may besuitably increased or decreased in consideration of their therapeuticeffects and safety.

Further, the composition of the present invention may contain otherpharmaceutically active ingredients, as far as they are not contrary tothe objects of the present invention.

The present invention will be described in more detail with reference tothe following examples, which is not intended to limit the presentinvention.

Example 1 Method

Test substance 1: 13,14-dihydro-15-keto-16,16-difluoro-prostaglandin E₁,which is a ClC-2 channel opener was used. The test substance 1 of 10 or100 μg/kg in 5 mL/kg of the vehicle was orally administered to maleWistar rats (six weeks old, weight: 180-210 g) that had been fasted forat least 16 hours. The control group received the same volume of thevehicle (0.5% ethanol and 0.01% polysorbate 80 in distilled water).Thirty minutes after the administration, the animals underwentlaparotomy under ether anesthesia. The first portion of the duodenum andend portion of the ileum were ligated respectively and the bowel wasremoved. The intestinal fluid of each animal was collected from theremoved bowel and the amount was determined. After the intestinal fluidwas centrifuged by 10,000×g for 5 minutes, its supernatant wascollected. The chloride ion concentration in the supernatant wasmeasured with a chloride counter (CL-7, Hiranuma Sangyo Co., Ltd.).Dunnett's test was used in comparing the control group and the testgroups receiving test substance 1 in each dose. P values less than 0.05were considered to be statistically significant.

(Result)

Table 1 shows the amount of the intestinal fluid and concentration ofchloride ion in the intestinal fluid in each group.

TABLE 1 Effect of Test Substance 1 on the Amount of Intestinal Fluid andConcentration of Chloride Ion in Intestinal Fluid in Rat Chloride IonConcentration In Amount of Intestinal Fluid Intestinal Mean ± S.E. GroupDose n Fluid mL mEq/L Control — 7 0.9 ± 0.1  41.8 ± 3.9 Test Sub. 1  10μg/kg 7 3.3 ± 0.3** 110.1 ± 5.6** Test Sub. 1 100 μg/kg 7 5.3 ± 0.2**126.6 ± 2.4** Dunnett's Test: Compared with the Control Group, **P <0.01

Administration of test substance 1 of 10 and 100μg/kg increased theamount of intestinal fluid and chloride ion concentration in theintestinal fluid in a dose-dependant manner. Additionally, compared withthe control group, there was a significant increase in the amount ofintestinal fluid and the concentration of chloride ion in intestinalfluid in each of the groups receiving test substance 1.

The above result suggests that a ClC-2 channel opener positivelyaccelerates the secretion of chloride ion in the bowel to increase theamount of intestinal fluid, and it is effective for treatment ofconstipation.

Example 2 Method

An eye drop composition comprising 0.001% of the test substance 1 wasinstilled to male white rabbit in the amount of 30 μL/eye. The controlgroup received the same amount of eye drop vehicle. Two hours after theinstillation, without anesthesia, one third of palpebra inferior (on theear side) was covered with the Schirmer's Paper (Showa Yakuhin KakoK.K., Japan, Lot No. 9011N) by binding its tip inside the conjunctivalsac for 1 minute. Then the amount of lacrimal fluid was measured fromthe length of wetting area on the paper filter with the scale on it.Lacrimal fluid 5 μL was collected from the conjunctival sac of palpebrainferior with a capillary pipet. The collected lacrimal fluid wasdiluted five fold with distilled water and chloride ion concentration inthe lacrimal fluid was measured with a chloride counter (CL-7, HiranumaSangyo Co. Ltd., Japan). Student's t-test and Wilcoxon's test were usedin comparing the control group and the group receiving test substance 1.P values less than 0.05 were considered to be statistically significant.

(Result)

Table 2 shows the amount of lacrimal fluid and the chloride ionconcentration in lacrimal fluid of each group.

TABLE 2 Effect of Test Substance 1 on the Amount of Lacrimal Fluid andthe Chloride Ion Concentration in Lacrimal Fluid in Rabbit Chloride IonConcentration in Amount of Lacrimal Fluid Lacrimal Mean ± S.E., GroupDose n Fluid mL mEq/L Control — 8  6.8 ± 0.5 116.9 ± 1.3 Test Sub. 10.001% 8 11.5 ± 0.7## 152.9 ± 7.7^((##)) Student's t-test: Compared withthe Control Group, ##P < 0.01 Wilcoxon's test: Compared with the ControlGroup, ^([##])P < 0.01

Compared with the control group, there was a significant increase in theamount of lacrimal fluid and chloride ion concentration in lacrimalfluid in the group receiving test substance 1.

The above result suggests that a ClC-2 channel opener positivelyaccelerates the secretion of chloride ion in the eye to increase theamount of lacrimal fluid, and it is effective for treatment of dry eye.

Example 3 Method

Test substance 2:13,14-dihydro-15-keto-16,16-difluoro-18(S)-methyl-prostaglandin E₁,which is a ClC-2 channel opener was used. Test substance 2 of 100 μg/kgwas orally administered to male Wistar rats (six weeks old, weight:180-210 g) three times a day for seven days. The control group receivedthe same amount of the vehicle (0.01% polysorbate 80 and 0.5% ethanol indistilled water). In the following morning of the final administrationday (about 17 hours after the final administration), a polyethylenecatheter (PE10, Becton Dickinson and Company) was inserted into therats' common bile duct under ether anesthesia. The rats were placed in aBorrmann's cage and were left for 1 hour to awake from anesthesia. Biledischarged during one hour from one to two hours after the insertion ofthe catheter was collected to measure the amount of bile. Chloride ionconcentration was measured with a chloride counter (CL-7, HiranumaSangyo Co., Ltd.). Student's t-test was used in comparing the controlgroup and the group receiving test substance 2. P values less than 0.05were considered to be statistically significant.

(Result)

Table 3 shows the amount of bile and chloride ion concentration in bilein each group.

TABLE 3 Effect of Test Substance 2 on the Amount of Bile and chlorideion concentration in Bile in Rats Chloride Ion Concentration in BileAmount of Mean ± S.E.,. Group Dose n Bile ML mEq/L Control — 7 364.4 ±26.1 91.1 ± 2.7 Test Sub. 2 100 μg/kg 8 491.9 ± 36.7* 98.4 ± 1.8**Student's t-test: Compared with the Control Group, *P < 0.05

Compared with the control group, there was a significant increase in theamount of bile and chloride ion concentration in bile in the groupreceiving test substance 2.

The above result suggests that a ClC-2 channel opener positivelyaccelerates the secretion of chloride ion in the liver to increase theamount of bile, and it is effective for treatment of hepatic disease aswell as hepatic transplantation.

Example 4 Method

Male Wistar rats were used. The common bile duct of both the test andthe control groups were catheterized to collect bile under pentobarbitalsodium anesthesia. After the catheter was inserted and fixed in theportal vein, Krebs-Ringer solution (pH 7.4, 37° C.) comprising sodiumtaurocolate (30 μmol/l) saturated with 95% 0₂ and 5% Co₂ was infused ata constant flow rate of 4.0 mL/min/g liver weight by means ofperistaltic pump. The liver was removed under perfusion. For the testgroup, test substance 2 was added to the Krebs-Ringer solution. Thirtyminutes after the perfusion, the amount of bile of both the test groupand the control group was measured. Chloride ion concentration in thebile was also measured. The amount of bile at 30 minutes after theadministration of test substance 2 or vehicle was shown as relativevalue (%) to the amount immediately before the administration.

(Result)

Table 4 shows the amount of bile and chloride ion concentration in bilein each group.

TABLE 4 Effect of Test Substance 2 on the Amount of Bile and chlorideion concentration in Bile in Rats Chloride Ion Relative AmountConcentration of Bile In Bile Concentration (%) μM/min/g liver Group(μm) n (Mean ± S.E) (Mean ± S.E.) Control — 5  92.4 ± 2.7 0.268 ± 0.007Test Sub. 2 10 5 126.2 ± 9.0** 0.353 ± 0.009** Compared with the ControlGroup, **P < 0.01 (one-way ANOVA with Fisher's multiple comparison test)

Compared with the control group, there was a significant increase in theamount of bile and chloride ion concentration in bile in the groupreceiving test substance 2.

The above result suggests that a ClC-2 channel opener positivelyaccelerates the secretion of chloride ion in the removed liver toincrease the amount of bile. This result suggests that ClC-2 channelopener is effective for treatment in organ transplantation procedureincluding hepatic transplantation.

Example 5 Method

Male Wistar rats (weight: about 280 g) were anesthetized withintraperitoneal injection of pentobarbital sodium (50 mg/kg) andhepatic-portal region was exposed by abdominal midline incision. Thecommon bile duct was cannulated and the portal stem was inserted with a19 gauge Surflo® needle equipped with a three way stopcock.Simultaneously, the liver was perfused with oxidized KrebsRinger Bufferat a rate of 4.0 mL/min/g liver and was bled by dissecting the inferiorvena cava. The liver was cut off from the tissues around it, removedoutside the body and subjected to extracorporeal perfusion. The liverwas subjected to a 15-minute pre-perfusion until its oxygen consumptionachieved to a constant state. Then the perfusate was replaced with anorgan preservative solution (University of Wisconsin solution; (U-Wsolution) at 4° C. Immediately after the replacement was completed, theliver connected with the three-way stopcock was removed from theperfusion cycle and the inlet was clamped. Then the liver was put in thepreservative solution (U-W solution) at the same temperature and storedfor 16 hours. After the 16-hour storage, the liver was reperfused withKrebs-Ringer Buffer. Thirty minutes after the reperfusion, the amount ofbile flow was measured.

(1) Effect of Substance 2 Added to the Organ Preservative Solution (U-WSolution)

In the above perfusion protocol, test substance 2 at the concentrationof 10 μM was added to the preservative solution (U-W solution).

(2) Effect of Substance 2 Added to the Perfusate During Reperfusion

In the above perfusion protocol, test substance 2 was not added to thepreservative solution, but added to the perfusate (Krebs-Ringer Buffer)for reperfusion at the concentration of 10 μM.

(Result)

Tables 5 and 6 respectively show the amount of bile flow at 30 minutesafter the reperfusion under the (1) and (2) conditions.

TABLE 5 Organ Preservative Effect of Test Substance 2 Added to thePreservative Solution Amount of Bile Flow, Concentration μL/min/g liverGroup (μM) n (Mean ± S.D.) Control — 5 2.11 ± 0.18 Test Sub. 2 10 5 2.43± 0.10* Compared with the Control Group, *P < 0.05

TABLE 6 Effect of Substance 2 Added to the Perfusate during ReperfusionAmount of Bile Flow, Concentration μL/min/g liver Group (μM) n (Mean ±S.D.) Control — 5 2.11 ± 0.18 Test Sub. 2 10 5 2.67 ± 0.31* Comparedwith the Control Group, *P < 0.05

Compared with the control group, there was a significant increase in theamount of bile flow in the group receiving substance 2, which was addedto the preservative solution and the perfusate during the reperfusion.

The above result suggests that a ClC-2 channel opener is effective forimproving preservation of organs, inhibiting disorders duringreperfusion and improving taking rate after transplantation, andtherefore, it is effective for treatment in organ transplantationprocedure including hepatic transplantation.

Example 6 Method

Test substance 1 of 0.3 mg/kg in 10 mL/kg of vehicle was subcutaneouslyadministered to ddY male mice (5 weeks old, weight: 27-30 g). Thecontrol group received the vehicle. Thirty minutes after theadministration, the mice were beheaded with guillotine. Duration ofgasping shown after beheading was measured.

(Result)

Table 7 shows the antianoxia effect in each group.

TABLE 7 Antianoxia Effect Dose Duration of Gasping Group (mg/kg) n (Mean± S.D.) Control — 10 20.3 ± 1.9 Test Sub. 1 0.3 10 24.1 :± 1.9** t-test:**p < 0.01

Compared to the control group, there was a significant increase in theduration of gasping in the group receiving test substance 1.

The above result suggests that a ClC-2 channel opener is effective fortreatment of atmospheric hypoxia, hypoxia or anoxia in the brain.

Example 7 Method

Test substance 1 of 0.1 or 0.01 mg/kg in 5 mL/kg of vehicle wassubcutaneously administered to male Wistar rats (7 weeks old, weight:200-250 g). The control group received the same amount of the vehicle(physiological Saline). Thirty minutes after the administration,ammonium sulfate 600 mg/mL was administered to the abdominal cavity ofthe rats. The survival rate at 30 minutes after the administration ofammonium sulfate was calculated. Animals that had survived until 30minutes after the administration of ammonium sulfate were killed underchloroform, and their lungs were removed to measure the weights.Regarding animals that had been dead by 30 minutes after theadministration of ammonium sulfate, their lungs were removed immediatelyafter their death to measure the weights. The lung-weight of therespective groups was compared to the lung-weight of the normal group,which received no ammonium sulfate. Based on the lung-weightmeasurements, the inhibitory rate of lung-weight increase of the groupreceiving test substance 1 as compared to that of the control group wascalculated.

(Result)

Table 8 shows the surviving rate and Table 9 shows the inhibitory rateof lung-weight increase.

TABLE 8 Antipneumonedema Effect (Surviving Rate) Dose Surviving RateGroup (mg/kg) n (%) Control — 10 30 Test Sub. 1 0.1 10 60

TABLE 9 Antipneumonedema Effect (Inhibitory Rate of Lung-weightIncrease) Inhibitory Rate of Lung- Dose weight Increase Group (mg/kg) n(%) Test Sub. 1 0.01 10 47

The above result suggests that a ClC-2 channel opener is effective fortreatment of lung disorder.

Example 8 Method

Effects on acute ulcer was determined by means of water-immersionrestraint stress induced ulcer model and indomethacin induced ulcermodel.

Test substance 1 was orally administered to male Wistar rats (weight:180-210 g) that had been fasted for 24 hours. In the water-immersionrestraint stress induced ulcer model, 10 minutes after the oraladministration of test substance 1, the rats were bound in a stress cageand immersed in water at 23° C. up to the level of their chestxiphisternums. Six hours after the immersion, the animals were killed bycervical dislocation. In the indomethacin induced ulcer model,immediately after the oral administration of test substance 1 or 2,indomethacin 10 mg/kg was orally administered. Five hours after theadministration, the animals were killed by cervical dislocation. Thestomachs removed from the animals were fixed with 1% formalin and thendissected along the greater curvatures. The major axis (mm) of eachulcer formed in the stomach was measured. The major axes of the ulcersin each animal were totaled, which were expressed as ulcer coefficients.Based on the ulcer coefficients of the control group and those of thegroups receiving test substances, the ulcer inhibitory rates werecalculated.

(Result)

Table 10 shows the anti-water restrictive stress ulcer effect and Table11 shows the anti-indomethacin ulcer effect.

TABLE 10 Anti-water' Restrictive Stress Ulcer Effect Ulcer InhibitoryDose Ulcer Coefficient Rate Group (ug/kg) n (Mean ± S.E.) (%) Control —10 29.3 ± 3.0 — Test Sub. 1  30 10 13.4 ± 2.0** 54.3 Test Sub. 1 100 10 4.3 ± 1.9** 85.3 Compared with the Control Group, **P < 0.01 (Dunnett'stest)

TABLE 11 Anti-indomethacin Ulcer Effect Ulcer Ulcer Inhibitory DoseCoefficient Rate Group (μg/kg) n (Mean ± S.E.) (%) Control — 10 49.6 ±7.6 — Test Sub. 1 2 10 27.9 ± 5.1 44   Control — 10 86.4 ± 9.6 — TestSub. 2 1 10 35.7 ± 8.3** 58.7 Test Sub. 2 3 10 30.0 ± 4 6** 65.3 TestSub. 2 10  10 19.7 ± 4.8** 77.2 Compared with the Control Group, **P <0.01 (Dunnett's test)

The above result suggests that a ClC-2 channel opener is effective fortreatment of gastric ulcer.

Example 9 Method

Mercuric chloride (HgC1₂) of 4 mg/kg was intramuscularly administered tomale Wistar rats (weight: 245-290 g) to induce ulcer in the largeintestine. Thirty minutes before, and 2 and 6 hours after theadministration of HgC1₂, test substance 2 was subcutaneouslyadministered. The control group received a vehicle. Twenty-four hoursafter the administration of HgC1₂, the animals were bled to death underether anesthesia. After abdominal incision, the upper portion of theileocecal opening and the upper portion of the anus were ligated, andthe large intestine region from the caecum to the rectum was removed.The removed large intestine was fixed with 1% formalin and thendissected, and the major and minor axes of ulcer were measured with acaliper under a stereoscopic microscope. The product of the major axismultiplied by the minor axis was expressed as the ulcer area. Further,the total ulcer area was calculated in each animal, which was expressedas the gross ulcer area.

(Result)

Table 12 shows the anti-large intestinal ulcer effect.

TABLE 12 Anti-large Intestinal Ulcer Effect Gross Ulcer Ulcer InhibitoryDose Area, mm² Rate Group (μg/kg) n (Mean ± S.E) (%) Control — 5 29.0 ±23.5 — Test Sub. 2  10 5 15.2 ± 15.8 54.3 Test Sub. 2 1000 5  3.7 ±8.3** 85.3 Compared with the Control Group, **P < 0.05 (Dunnett's test)

The above result suggests that a ClC-2 channel opener is effective fortreatment of ulcer in the bowel.

Example 10 Method

Male Wistar rats (weight: 240-270 g) that had been fasted overnight weresubjected to abdominal incision under pentobarbital sodium anesthesia.Then a mixed solution of 10% taurocholic acid 3 mL and 0.3% trypsinsolution 0.3 mL was antidroicly infused from the bile duct duodenumopening to induce pancreatitis. Thirty minutes before, and 2 and 4 hoursafter the infusion of the mixed solution of taurocholic acid andtrypsin, test substance 2 was subcutaneously administered. The controlgroup received the vehicle. Six hours after the infusion of the mixedsolution of taurocholic acid and trypsin, the blood was collected tomeasure the serum amylase activity.

(Result)

Table 13 shows the anti-pancreatitis effect.

TABLE 13 Anti-pancreatitis Effect serum amylase, Dose IU/L (Mean ±Inhibitory Rate Group (μg/kg) n S.E.) (%) Control — 12 3141 ± 838 — TestSub. 2 100 9 2430 ± 417 22.6

The above result suggests that a ClC-2 channel opener is effective fortreatment of pancreatitis.

Example 11 Method

Beagle dogs (weight: 8.8-10.3 kg) were used, and under thiopental sodiumand α-chloralose anesthesia, their cardiac output was guided from a FJprobe equipped on the aortic arch via a rectangular wave electromagneticblood flow meter WFV-3100, Nihon Koden) to a blood flow indicating unitand recorded in a recorder. The model of cardiac incompetence wasprepared in accordance with the following method. A continuous infusionof lactate Ringer solution (5 mL/body/min) having dissolved therein DLpropranolol (0.06 mg/mL) from a catheter inserted into the left femoralvein started diastolic overload. Thirty minutes later, the left anteriordescendence (LAD) was ligated. Thirty minutes after ligating the LAD,methoxamine solution was continuously infused from the right femoralvein (5 μg/kg/min), and the infusion of methoxamine solution wasincreased by 5 μg/kg/min until the cardiac output was reduced by atleast 20% from that before the diastolic overload, and the infusion wasmaximally increased by 20 ug/kg/min. At the time the cardiac output wasreduced by at least 20% from that before the diastolic overload, thedose of methoxamine was reduced to 5 μg/kg/min and the infusion oflactate Ringer solution was reduced to 3 mL/kg/min, which was continueduntil the completion of experiments. Sixty to 90 minutes after thecardiac output was reduced by at least 20% and other hemodynamicparameters were stabled, test substance 2 was administered via a cannulaindwelled in the duodenum. The control group received a vehicle. Theratio of the cardiac output at 30 minutes after the administration oftest substance 2 as compared to the cardiac output immediately beforethe administration of test substance 2 or a vehicle was expressed by arelative value (%).

(Result)

Table 14 shows the cardiac output increasing effect.

TABLE 14 Cardiac Output Increasing Effect Relative Cardiac Dose Output,% Group (μg/kg) n (Mean ± S.E.) Control — 15  97.7 ± 2.13 Test Sub 2 10015 108.7 ± 0.47* Test Sub. 2 300 15 117.8 ± 2.42** Compared with theControl Group, *P < 0.05, **P < 0.01 (Dunnett's test)

The above result suggests that a ClC-2 channel opener is effective fortreatment of cardiovascular system disorder.

1. A method for prophylactic or therapeutic treatment of a condition ordisease responsive to opening of ClC-2 channel, which comprises the stepof administering an effective amount of a ClC-2 channel opener to asubject in need of said treatment, wherein said disease is associatedwith abnormality in the secretion of fluid.
 2. The method as describedin claim 1, wherein said disease is constipation.